Csaba Cséke

Regulation of the formation and utilization of photosynthate in leaves

II. Regulation of carbon transformations by chloroplasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 A. The reductive pentose phosphate cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 1. The ferredoxin/thioredoxin system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 2. The NADP/thioredoxin system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 3. Coordinate regulation of photosynthetic enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 B. Starch synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 C. Starch degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

An enzyme synthesizing fructose 2,6-bisphosphate occurs in leaves and is regulated by metabolite effectors

An enzyme catalyzing the ATP and fructose 6‐phosphate‐dependent synthesis of fructose 2,6‐bisphosphate, a regulator of glycolysis and gluconeogenesis, has been identified and partially purified from plants, specifically the cytoplasmic fraction of spinach leaf parenchyma cells. The enzyme, designated fructose 6‐phosphate, 2‐kinase, showed no response to a protein phosphorylation system known to inhibit the corresponding enzyme in mammalian cells, but it responded strikingly to metabolite effectors (Pi, an activator/PGA, an inhibitor) through changes in substrate affinity and maximal velocity. The observed pattern of regulation suggests a role for chloroplasts in controlling cytoplasmic carbon processing.

Lack of Cross-Resistance of Imazaquin-Resistant Xanthium strumarium Acetolactate Synthase to Flumetsulam and Chlorimuron.

Acetolactate synthase (ALS) was isolated from a field population of cocklebur (Xanthium strumarium) that developed resistance to the herbicide Scepter following three consecutive years of application. The active ingredient of Scepter, imazaquin, gave an inhibitor concentration required to produce 50% inhibition of the enzyme activity that was more than 300 times greater for the resistant enzyme than for the wild-type cocklebur ALS. Tests with flumetsulam and chlorimuron show that the resistant ALS was not cross-resistant to these two other classes of ALS inhibitors.

Metabolite-mediated catalyst conversion of PFK and PFP: a mechanism of enyme regulation in green plants

Metabolites known to occur in the cytosol of photosynthetic leaf cells were found to mediate the reversible conversion of pyrophosphate—D‐fructose‐6‐phosphate 1‐phosphotransferase (PFP) to phosphofructokinase (PFK) in partially purified preparations from spinach leaves. Preincubation of PFP with fructose 2,6‐bisphosphate, ATP or fructose 6‐phosphate converted PFP to PFK. The reverse reaction (PFK → PFP) was promoted by UDP‐glucose plus pyrophosphate. These conversions in catalytic capability were accompanied by changes in molecular mass and charge. The results are in accord with the view that the alterations in PFP and PFK activity, provisionally called ‘metabolite‐mediated catalyst conversion’, represent a regulatory mechanism to direct left cytosolic carbon flux in either the biosynthetic or degradatory direction.

A product‐regulated fructose 2,6‐bisphosphatase occurs in green leaves

An enzyme catalyzing the hydrolytic conversion of fructose 2,6‐bisphosphate (Fru‐2,6‐P2) to fructose 6‐phosphate (Fru‐6‐P) and Pi has been identified and purified from plants, specifically the cytosolic fraction of spinach leaf parenchyma cells. Partially purified preparations of the enzyme, designated fructose 2,6‐bisphosphatase (Fru‐2,6‐P2ase), were inhibited by products of the reaction (i.e., Pi and Fru‐6‐P) but showed no response to a protein phosphorylation system known to inhibit the corresponding enzyme in mammalian cells. Fru‐2,6‐P2ase co‐purified with fructose 6‐phosphate,2‐kinase, the enzyme catalyzing the synthesis of Fru‐2,6‐P2. The observed pattern of regulation of the enzymes functional in the synthesis and breakdown of Fru‐2,6‐P2 reinforces the conclusion that chloroplasts play a role in controlling cytosolic carbon processing in leaves.

Localization of NADP-malate dehydrogenase of corn leaves by immunological methods

Abstract An antibody raised against the inactive (non-activated) form of NADP-linked malate dehydrogenase (MDH) from corn leaves precipitated the enzyme and inhibited its catalytic activity. The antibody had no significant effect on the low levels of NAD-linked activity in the preparation. When applied in enzyme-linked immunosorption assay (ELISA) and immunofluorescence procedures, the antibody revealed that NADP-MDH is located exclusively in chloroplasts of mesophyll cells. The evidence also indicated that NADP-MDH undergoes a conformational change when activated. The results demonstrate that immunological methods can be used to determine the cellular location of C4 enzymes both in vitro and in situ.

Regulation of photosynthetic sucrose synthesis by fructose 2,6-bisphosphate

Fructose 2,6-bisphosphate (F2,6P2) is a signal metabolite, first found in liver where it has an important role in controlling glycolysis and gluconeogensis. F2,6P2 is present in spinach leaves (Cseke et al., 1982) where it is located in the cytosol with concentrations of 5–20 μM (Stitt et al., 1983b). The cytosolic fructose 1,6-bisphosphatase (FBPase) is inhibited by F2,6P2 (Cseke et al., 1982, Stitt et al., 1982). More detailed studies (see these Proceedings, Heldt et al.) have shown that F2,6P2 alters the cytosolic FBPase from hyperbolic substrate dependence (Km FBP = 3 μM) to sigmoidal dependence with a much lower substrate affinity (S 0.5 FBP = 0.1 and 0.25 mM in the presence of 2 and 10 μM F2,6P2 respectively. This means that the cytosolic FBPase, which catalyses the first irreversible step in the conversion of trioseP to sucrose in the cytosol, would be particularly sensitive to control by alterations in the concentrations of F2,6P2 and FBP.

Fructose-2,6-Bisphosphate: A Regulatory Metabolite Directing Carbon Flow between Sucrose and Starch

Evidence accumulated in the past few years suggests that fructose-2, 6-bisphosphate (Fru-2,6-P2), a regulatory metabolite localized in the cytosol of plant cells, can play a central role in the coordination of chloroplast and cytosolic carbohydrate metabolism of leaves (1). In the chloroplast the principal and ultimate regulator of carbohydrate metabolism is light. Light, absorbed by chlorophyll, is converted to different regulatory signals, which modulate selected enzymes in the chloroplast. Modification of sulphhydryl status of selected enzymes via the ferredoxin/ thioredoxin system and modulation of enzymes by light induced changes in the concentration of certain ions and metabolites are the major light driven regulatory mechanisms (2). (Fig. 1.)

The Regulation of Enzymes of Sucrose Metabolism in Plant Sinks

The regulation of sucrose breakdown and synthesis and the partitioning of carbon in sink tissues is not well understood. We present here a summary of what has been learned about these processes in both source and sink tissues and describe how our recent results add to our understanding of carbon metabolism in plant tissues.